I. Field of the Invention
The present invention relates generally to the fields of virology and genetic therapy. More particularly, it concerns coexpressing envelope proteins for producing a high titer pseudotyped viral composition.
II. Description of Related Art
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer of sheep called ovine pulmonary carcinoma or sheep pulmonary adenomatosis (Palmarini et al., 1999). Tumors originate from type II secretory alveolar and nonciliated bronchiolar epithelial cells, and late stages of the disease are accompanied by the secretion of copious lung fluid containing the virus. Purified virus induces multifocal tumors in as little as 10 days (Sharp et al., 1983). JSRV is a simple retrovirus with typical gag, pol, and env genes. The JSRV envelope glycoprotein appears to have transforming properties when it is expressed in certain cell types. The viral structural (gag) and enzymatic (pol) proteins interact primarily with viral components. The envelope (env) protein interacts with cellular components to mediate virus entry. The contagious nature of JSRV and its ability to survive exposure to proteases and surfactants present in lung fluid suggest that vectors based on JSRV might be useful for gene therapy, particularly therapies targeted to the lung, provided that the pathogenic features of the virus can be controlled.
Viral vectors can transduce genes into target cells with high efficiencies via specific virus envelope-host cell receptor interaction and viral mechanisms for gene expression. Consequently, viral vectors have been used as vehicles for the transfer of genes into many different cell types including whole embryos, fertilized eggs, isolated tissue samples, and cultured cell lines. The ability to introduce and express a foreign gene in a cell is useful for the study of gene expression and the elucidation of cell lineages (Watson et al., 1992). Retroviral vectors, capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (Watson et al., 1992). Viral vectors, and retroviral vectors in particular, are also used in therapeutic applications (e.g., gene therapy), in which a gene (or genes) is added to a cell to replace a missing or defective gene, or to provide a therapeutic gene not normally expressed in the infected cell.
In view of the wide variety of potential genes available for therapy, it is clear that an efficient means of delivering these genes is sorely needed in order to fulfill the promise of gene therapy for treating infectious, as well as non-infectious diseases. Several viral systems including murine retrovirus, lentivirus, adenovirus, parvovirus (adeno-associated virus), vaccinia virus, and herpes virus have been developed as therapeutic gene transfer vectors (For review see, Nienhuis et al., 1993).
Factors affecting viral vector usage include tissue tropism, stability of virus preparations, genome packaging capacity, and construct-dependent vector stability. In addition, in vivo application of viral vectors is often limited by host immune responses against viral structural proteins and/or transduced gene products.
The low production of recombinant virus produced by some retroviral system (e.g., 106 transducing units (tu)/ml or lower) compared to the adenoviral system (up to 1012 particles/ml) means that human cells are infected at a very low efficiency. This low efficiency is particularly problematic when the target cell type is represented at very low numbers in the tissue to be infected. The hematopoietic stem cell is a preferred target for gene therapy in a large number of disorders, these cells are present at very low frequencies. For example, totipotent embryonic stem cells have been reported to occur at a frequency of 10−4 to 10−6 in bone marrow (Glick and Pasternak, 1994). Thus, the low titer produced by existing vector systems is highly problematic for stem cell infection, as well as other therapies.
Additional vector systems are needed to provide a means of delivering and expressing genes efficiently in mammalian cells, particularly human cells. Various new methods and compositions are necessary if the promise of gene therapy is to be realized.